Peptide-polymer conjugates

ABSTRACT

This invention relates to a conjugate of a polymer moiety and an erythropoietin moiety.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.12/533,147, filed on Jul. 31, 2009, which claims the benefit of U.S.Provisional Application No. 61/085,072 filed on Jul. 31, 2008. Thecontents of both prior applications are hereby incorporated by referencein their entirety.

BACKGROUND

Advance in cell biology and recombinant protein technologies has led tothe development of protein therapeutics.

Yet, major hurdles still exist. Most proteins are susceptible toproteolytic degradation and therefore have a short circulating time.Other disadvantages include low water solubility and inducement ofneutralizing antibodies.

Attachment of a polymer, e.g., polyethylene glycol (PEG), to a proteinhinders access of proteolytic enzymes to the protein backbone, resultingin enhanced protein stability. In addition, it may also improve watersolubility and minimize immuogenicity. There is a need for effectivemethods of attaching polymer to proteins.

SUMMARY

An aspect of the present invention relates to polymer-polypeptideconjugates of formula I:

wherein each of R₁, R₂, R₃, R₄, and R₅, independently, is H, C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteraryl, C₃₋₈ cycloalkyl,or C₃₋₈ heterocycloalkyl; each of A₁ and A₂, independently, is a polymermoiety (e.g., a polyalkylene oxide moiety); each of G₁, G₂, and G₃,independently, is a bond or a linking functional group; P is aninterferon-β (INF-β) moiety, an erythropoietin (EPO) moiety, or a growthhormone (GH) moiety; m is 0 or an integer of 1-10; and n is an integerof 1-10. In these conjugates, the N-terminus of the INF-β moiety, theEPO moiety, or the GH moiety is bonded to G₃.

Referring to the above formula, the polymer-polypeptide conjugates haveone or more of the following features: A₁ and A₂ are polyalkylene oxidemoieties having a molecular weight of 2-100 kD (preferably 10-30 kD,e.g., 20 kD); each of G₁ and G₂ is

(in which the O atom is bonded to A₁ or A₂, and the N atom is bonded toa carbon atom as shown in formula I; each of G₁ and G₂ is urea,sulfonamide, or amide (in which the N atom is bonded to a carbon atom asshown in formula I); m is 4; n is 2; and each of R₁, R₂, R₃, R₄, and R₅is H. In some of these conjugates, P is rINF-β Ser₁₇ or a modified INF-βmoiety containing 1-4 additional amino acid residues at the N-terminusof the INF-β.

Another aspect of the present invention relates to polymer-peptideconjugates of formula II:

A-G₁-L-G₂-P,   formula II

wherein A is a polymer moiety (e.g., a polyalkylene oxide moiety); eachof G₁ and G₂, independently, is a bond or a linking functional group; Lis C₂₋₁₀ alkenylene or C₂₋₁₀ alkynylene; and P is an INF-β moiety, anEPO moiety, or a GH moiety. In these conjugates, the N-terminus of theINF-β moiety, the EPO moiety, or the GH moiety is attached to G₂.

Referring to formula II, the polymer-peptide conjugates have one or moreof the following features: A₁ and A₂ are polyalkylene oxide moietieshaving a molecular weight of 2-100 kD (preferably 10-30 kD, e.g., 20kD), each of G₁ and G₂ is a bond, C₆ is alkenylene, and each of R₁, R₂,R₃, R₄, and R₅ is H.

Another aspect of the present invention relates to polymer-peptideconjugates of

wherein each of R₁, R₂, R₃, and R₄, independently, is H, C₂₋₁₀ alkenyl,C₂₋₁₀ alkynyl, aryl, heteraryl, C₃₋₈ cycloalkyl, or C₃₋₈heterocycloalkyl; n is an integer of 2-10; A is a polymer moiety; G is alinking functional group; and P is a peptide moiety, the nitrogen atomof the N-terminus of the peptide moiety being bonded to the carbon atomin the

moiety shown in the formula above.

Referring to formula II, the polymer-peptide conjugates have one or moreof the following features: n is 1; A is polyalkylene oxide moietieshaving a molecular weight of 10-40 kD or 20-30 kD; G is

in which the O atom is bonded to A, and the N atom is bonded to a carbonatom; and P is an INF moiety, an EPO moiety, a GH moiety.

The term “C₁₋₁₀ alkyl” used herein refers to a straight-chained orbranched hydrocarbon mono-valent radical containing 1 to 10 carbonatoms. Examples of alkyl groups include methyl, ethyl, n-propyl,isopropyl, tert-butyl, and n-pentyl. Similarly, the term “C₂₋₁₀ alkenyl”refers to a straight-chained or branched hydrocarbon mono-valent radicalcontaining 2 to 10 carbon atoms and one or more double bonds. The term“C₂₋₁₀ alkynyl” refers to a straight-chained or branched hydrocarbonmono-valent radical containing 2 to 10 carbon atoms and one or moretriple bonds. The term “C₂₋₁₀ alkenylene” refers to a straight-chainedor branched hydrocarbon bi-valent radical containing 2 to 10 carbonatoms and one or more double bonds. The term “C₂₋₁₀ alkynylene” refersto a straight-chained or branched hydrocarbon bi-valent radicalcontaining 2 to 10 carbon atoms and one or more triple bonds.

The term “aryl” used herein refers to a hydrocarbon ring system(mono-cyclic or bi-cyclic) having at least one aromatic ring. Examplesof aryl moieties include, but are not limited to, phenyl, naphthyl, andpyrenyl.

The term “heteroaryl” used herein refers to a hydrocarbon ring system(mono-cyclic or bi-cyclic) having at least one aromatic ring whichcontains at least one heteroatom such as O, N, or S as part of the ringsystem and the reminder being carbon. Examples of heteroaryl moietiesinclude, but are not limited to, furyl, pyrrolyl, thienyl, oxazolyl,imidazolyl, thiazolyl, pyridinyl, pyrimidinyl, quinazolinyl, andindolyl.

The term “cycloalkyl” used herein refers to a partially or fullysaturated mono-cyclic or bi-cyclic ring system having only carbon ringatoms. Examples include, but are not limited to, cyclopropanyl,cyclopentanyl, and cyclohexanyl.

The term “heterocycloalkyl” used herein refers to a partially or fullysaturated mono-cyclic or bi-cyclic ring system having, in addition tocarbon, one or more heteroatoms (e.g., O, N, or S), as ring atoms.Examples include, but are not limited to, piperidine, piperazine,morpholine, thiomorpholine, and 1,4-oxazepane.

Alkyl, alkenyl, alkynyl, alkenylene, alkynylene, aryl, heteroaryl,cycloalkyl, and heterocycloalkyl mentioned herein include bothsubstituted and unsubstituted moieties. Examples of substituents includeC₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₈ cycloalkyl, C₅-C₈cycloalkenyl, C₁-C₁₀ alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy,amino, C₁-C₁₀ alkylamino, C₁-C₂₀ dialkylamino, arylamino, diarylamino,hydroxyamino, alkoxyamino, C₁-C₁₀ alkylsulfonamide, arylsulfonamide,hydroxy, halogen, thio, C₁-C₁₀ alkylthio, arylthio, cyano, nitro, acyl,acyloxy, carboxyl, and carboxylic ester.

The term “polymer moiety” refers to a mono-valent radical derived fromlinear, branched, or star-shaped polymer. The molecular weight of thepolymer moiety may be 2-100 kD. Examples of the polymer moiety include,but are not limited to, polyethylene oxide, polyethylene glycol,polyisopropylene oxide, polybutenylene oxide, polyethylene glycol, andcopolymers thereof. Other polymers such as dextran, polyvinyl alcohols,polyacrylamides, or carbohydrate-based polymers can also be used as longas they are not antigenic, toxic, or eliciting immune response.

The term “polypeptide moiety” refers to a mono-valent radical derivedfrom either a naturally occurring polypeptide or a modified polypeptide.The naturally occurring peptide can be INF-α_(2b), INF-β, GH, EPO, andgranulocyte colon-stimulating factor, or antibody. The modified peptidecan be, e.g., a peptide containing INF and 1-4 additional amino acidresidues at the N-terminus of the INF-α_(2b). An example of such amodified INF is

IFN representing an INF-α_(2b) moiety, the amino group at the N-terminusof which is bonded to the carbonyl group.

The term “interferon-β” refers to a family of highly homologous proteinsthat inhibit viral replication and cellular proliferation and modulateimmune response. See Derynck et al., (1980). Nature 285 (5766): 542-7;and Taniguchi et al., (1980). Gene 10 (1): 11-5. It includes bothnaturally occurring INF-βs and their functional equivalents, i.e., apolypeptide having at least 80% (e.g., 85%, 90%, 95%, or 99%) identicalto its wild-type counterpart. Examples of INF-βinclude the activeingredients in the commercially available drugs, such as Avonex,Betaseron, and Rebif. See, e.g., Etemadifar M. et al., Acta Neurol.Scand., 2006, 113(5): 283-7.

Listed below are amino acid sequences of exemplary human INF-β proteins,either in precursor form or in mature form:

mtnkcllqia lllcfsttal smsynllgfl qrssnfqcqk llwqlngrle yclkdrmnfd ipeeikqlqq fqkedaalti yemlqnifai frqdssstgw netivenlla nvyhqinhlk tvleekleke dftrgklmss lhlkryygri lhylkakeyshcawtivrve ilrnfyfinr ltgylrn

(See GenBank Accession No.: M28622, the Apr. 27, 1993 version;italicized portion refers to the signal peptide)

mnsfstsafg pvafslglll vlpaafpapv ppgedskdvaaphrqpltss eridkqiryi ldgisalrke tcnksnmcesskealaennl nlpkmaekdg cfqsgfneet clvkiitgllefevyleylq nrfesseeqa ravqmstkvl iqflqkkaknldaittpdpt tnaslltklq aqnqwlqdmt thlilrsfke flqsslralr qm

(See GenBank Accession No.: CAA00839, the Dec. 3, 1993 version)

In one example, the INF-β is mutant rINF-β Ser₁₇ (recombinant INF-β, inwhich serine is in place of cysteine at position 17 in the native matureINF-β sequence). The amino acid of this mutant is shown below:

synllgflqr ssnfqsqkll wqlngrleyc lkdrmnfdipeeikqlqqfq kedaaltiye mlqnifaifr qdssstgwnetivenllanv yhqinhlktv leeklekedf trgklmsslhlkryygrilh ylkakeyshc awtivrveil rnfyfinrlt gylrn

In another example, the INF-β is a modified native INF-β, in which 1-4additional amino acid residues are attached to the N-terminus of thenative INF-β.

EPO, produced by either liver or kidney, is a glycoprotein hormone thatcontrols erythropoiesis or red blood cell production. It includes bothnaturally occurring EPO and its functional equivalents. See U.S. Pat.No. 5,621,080 and US Patent Application Publication 20050176627. Theamino acid sequences of human EPO (in precursor and mature form) areshown below:

mgvhecpawl wllls11slp lglpvlgapp rlicdsrvlerylleakeae nittgcaehc slnenitvpd tkvnfyawkrmevgqqavev wqglallsea vlrgqallvn ssqpweplqlhvdkavsglr slttllralg aqkeaisppd aasaaplrtitadtfrklfr vysnflrgkl klytgeacrt gdr (precursor)apprlicdsr vlerylleak eaenittgca ehcslnenitvpdtkvnfya wkrmevgqqa vevwqglall seavlrgqallvnssqpwep lqlhvdkavs glrslttllr algaqkeaisppdaasaapl rtitadtfrk lfrvysnflr gklklytgea crtgdr (mature form)

An EPO protein used to make the conjugate of this invention can be anEPO protein, either in precursor or mature form, produced by a suitablespecies, e.g., human, murine, swine, or bovine. In one example, the EPOprotein has an amino acid sequence at least 80% (e.g., 85%, 90%, 95% or99%) identical to one of the amino acid sequences shown above. Inanother example, the EPO is a modified native EPO in which 1-4additional amino acid residues are attached to the N-terminus of thenative EPO.

The term “growth hormone” refers to the naturally occurring human growthhormone, either in precursor or mature form, and its functionalvariants, i.e., having an amino acid sequence at least 80% (e.g., 85%,90%, 95%, or 99%) identical to the naturally occurring human growthhormone and possessing the same physiological activity of that humangrowth hormone. In one example, the growth hormone is a modified nativegrowth hormone in which 1-4 additional amino acid residues are attachedto the N-terminus of the native growth hormone. The amino acid sequencesof the naturally occurring human growth hormone (in precursor and matureform) are shown below:

matgsrtsll lafgllclpw lqegsafpti plsrlfdnamlrahrlhqla fdtyqefeea yipkeqkysf lqnpqtslcfsesiptpsnr eetqqksnle llrisllliq swlepvqflrsvfanslvyg asdsnvydll kdleegiqtl mgrledgsprtgqifkqtys kfdtnshndd allknyglly cfrkdmdkve tflrivqcrs vegscgf(precursor) fptiplsrlf dnamlrahrl hqlafdtyqe feeayipkeqkysflqnpqt slcfsesipt psnreetqqk snlellrisllliqswlepv qflrsvfans lvygasdsnv ydllkdleegiqtlmgrled gsprtgqifk qtyskfdtns hnddallknygllycfrkdm dkvetflriv qcrsvegscg f (mature form)mfptiplsrl fdnamlrahr lhqlafdtyq efeeayipkeqkysflqnpq tslcfsesip tpsnreetqq ksnlellrisllliqswlep vqflrsvfan slvygasdsn vydllkdleegiqtlmgrle dgsprtgqif kqtyskfdtn shnddallknygllycfrkd mdkvetflri vqcrsvegsc gf (modified form)

The “percent identity” of two amino acid sequences is determined usingthe algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad.Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into theNBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol.Biol. 215:403-10, 1990. BLAST protein searches can be performed with theXBLAST program, score=50, wordlength=3 to obtain amino acid sequenceshomologous to the protein molecules for use in the invention. Where gapsexist between two sequences, Gapped BLAST can be utilized as describedin Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

The term “linking functional group” refers to a bi-valent functionalgroup, one end being connected to the polymer moiety and the other endbeing connected to the peptide moiety. Examples include, but are notlimited to, —O—, —S—, carboxylic ester, carbonyl, carbonate, amide,carbamate, urea, sulfonyl, sulfinyl, amino, imino, hydroxyamino,phosphonate, or phosphate group.

The peptide-polymer conjugate described above can be in the free form orin the form of salt, if applicable. A salt, for example, can be formedbetween an anion and a positively charged group (e.g., amino) on apeptide-polymer conjugate of this invention. Suitable anions includechloride, bromide, iodide, sulfate, nitrate, phosphate, citrate,methanesulfonate, trifluoroacetate, and acetate. Likewise, a salt canalso be formed between a cation and a negatively charged group (e.g.,carboxylate) on a polypeptide-polymer conjugate of this invention.Suitable cations include sodium ion, potassium ion, magnesium ion,calcium ion, and an ammonium cation such as tetramethylammonium ion.

In addition, the peptide-polymer conjugate may have one or more doublebonds, or one or more asymmetric centers. Such a conjugate can occur asracemates, racemic mixtures, single enantiomers, individualdiastereomers, diastereomeric mixtures, and cis- or trans- or E- orZ-double bond isomeric forms.

Examples the polymer-peptide conjugate of this invention is shown below:

in which mPEG represents methoxy-capped polyethylene glycol having amolecular weight of 20 kD, and the N-termini of rINF-βSer₁₇, EPO, and GHare attached to the rightmost carbon shown in the above structures.

Certain proteins have therapeutic utilities. The conjugates of thisinvention, containing a peptide moiety, can therefore be used to treatdisease. For example, INF-β is an immunomodulating medication fortreating HCV or HBV infection. See, e.g., Journal of Vascular andInterventional Radiology 13 (2002): 191-196. Thus, within the scope ofthis invention is a method of treating hepatitis C virus (HCV) infectionor hepatitis B virus (HBV) infection with an INF-β-polymer conjugatedescribed above. As another example, EPO is a hormone produced by thekidney to promote the formation of red blood cells in the bone marrow.It has been used as an immunomodulating medication for treating anaemiaresulting from chronic kidney disease, anemia secondary to zidovudinetreatment of AIDS, and anemia associated with cancer. Recent studieshave also found that EPO enhances neurogenesis and plays a critical rolein post-stroke recovery. See, e.g., P. T. Tsai, Journal of Neuroscience,2006, 26: 1269. Thus, another aspect of this invention relates to amethod of treating aneamia or enhancing neurogenesis by an EPO-polymerconjugate described above.

Also within the scope of this invention is a composition containing theINF-β-polymer conjugate described above for use in treating HCVinfection or HBV infection, and a composition containing the EPO-polymerconjugate described above for use in treating aneamia or enhancingneurogenesis, as well as the therapeutic use and use of the conjugatefor the manufacture of a medicament for treating HCV infection, HBVinfection, or aneamia, or for enhancing neurogenesis.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and from the claims.

DETAILED DESCRIPTION

The peptide-polymer conjugates of the present invention can be preparedby synthetic methods well known in the chemical art. For example, onecan combine a linker molecule having one or more active functionalgroups with two polymer molecules having a functional group reactive tothose on the linker molecule. Subsequently, a peptide moleculecontaining a functional group is reacted with a functional group of thelinker molecule to form a peptide-polymer conjugate of this invention.Two illustrative synthetic schemes are provided herein.

Scheme 1 below shows an example of preparing the peptide-polymerconjugates of formula I. Diamine compound 1, which contains an acetalgroup, is reacted with N-hydroxysuccinimidyl carbonate mPEG (i.e.,compound 2) to form di-PEGylated compound 3, which is subsequentlyconverted to aldehyde 4. This aldehyde compound is reacted with peptideH-P having a free amino group via reductive alkylation to afford apeptide-polymer conjugate of this invention.

Scheme 2 below shows an example of preparing the peptide-polymerconjugates of formula II. Chemical 6 has a polymer moiety and analdehyde functional group. It can be reacted with peptide 7, which has afree amino functional group. The resulting product 8 is subsequentlyreduced, e.g., by hydrogenation or by NaBH₃CN, to afford peptide-polymerconjugate 9.

Scheme 3 below is an example of preparing a peptide-polymer conjugate offormula III. Compound 10 having an acetal group, which can be preparedfrom β-amino acid, is reacted with N-hydroxysuccinimidyl carbonate mPEG2 to form PEGylated compound 11, which is subsequently converted toaldehyde 12. This aldehyde compound is reacted with peptide H-P having afree amino group via reductive alkylation to afford desired compound 13.

The chemical reactions described above include using solvents, reagents,catalysts, protecting group and deprotecting group reagents, and certainreaction conditions. They may additionally include steps, either beforeor after the steps described specifically herein, to add or removesuitable protecting groups in order to ultimately allow for synthesis ofa peptide-polymer conjugate. In addition, various synthetic steps may beperformed in an alternate sequence or order to give the desiredpolypeptide-polymer conjugates. Synthetic chemistry transformations andprotecting group methodologies (protection and deprotection) useful insynthesizing applicable peptide-polymer conjugates are known in the artand include, for example, those described in R. Larock, ComprehensiveOrganic Transformations, VCH Publishers (1989); T. W. Greene and P. G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley andSons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995) and subsequent editions thereof.

A peptide-polymer conjugate thus synthesized can be further purified bya method such as ion exchange chromatography, gel filtrationchromatography, electrophoresis, dialysis, ultrafiltration, orultracentrifugation.

The peptide-polymer conjugate of the invention may be pharmaceuticallyactive in the conjugate form. Alternatively, it can release apharmaceutically active peptide in vivo (e.g., through hydrolysis) byenzymatically cleaving the linkage between the peptide moiety and thepolymer moiety. Examples of enzymes involved in in vivo cleavinglinkages include oxidative enzymes (e.g., peroxidases, amine oxidases,or dehydrogenases), reductive enzymes (e.g., keto reductases), andhydrolytic enzymes (e.g., proteases, esterases, sulfatases, orphosphatases).

Thus, one aspect of this invention relates to a method of administeringan effective amount of one or more of the above-describedpeptide-polymer conjugates for treating a disorder (e.g., HCV or HBVinfection, or aneamia). Specifically, a disease can be treated byadministering to a subject one or more of the peptide-polymer conjugatesin an effective amount. Such a subject can be identified by a healthcare professional based on results from any suitable diagnostic method.

As used herein, the term “treating” or “treatment” is defined as theapplication or administration of a composition including apeptide-polymer conjugate to a subject (human or animal), who has adisorder, a symptom of the disorder, a disease or disorder secondary tothe disorder, or a predisposition toward the disorder, with the purposeto cure, alleviate, relieve, remedy, or ameliorate the disorder, thesymptom of the disorder, the disease or disorder secondary to thedisorder, or the predisposition toward the disorder. “An effectiveamount” refers to an amount of a peptide-polymer conjugate which confersa therapeutic effect on the treated subject. The therapeutic effect maybe objective (i.e., measurably by some tests or markers) or subjective(i.e., a subject gives an indication of or feels an effect).

To practice the method of the present invention, a composition havingone or more of the above-mentioned conjugates can be administeredparenterally, orally, nasally, rectally, topically, or buccally. Theterm “parenteral” as used herein refers to subcutaneous, intracutaneous,intravenous, intramuscular, intraarticular, intraarterial,intrasynovial, intrasternal, intrathecal, intralesional,intraperitoneal, intratracheal or intracranial injection, as well as anysuitable infusion technique.

A sterile injectable composition can be a solution or suspension in anon-toxic parenterally acceptable diluent or solvent, such as a solutionin 1,3-butanediol. Among the acceptable vehicles and solvents that canbe employed are mannitol, water, Ringer's solution, and isotonic sodiumchloride solution. In addition, fixed oils are conventionally employedas a solvent or suspending medium (e.g., synthetic mono- ordi-glycerides). Fatty acid, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions can also contain a long chain alcohol diluent or dispersant,or carboxymethyl cellulose or similar dispersing agents. Other commonlyused surfactants such as Tweens or Spans or other similar emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms can also be used for the purpose of formulation.

A composition for oral administration can be any orally acceptabledosage form including capsules, tablets, emulsions, and aqueoussuspensions, dispersions, and solutions. In the case of tablets,commonly used carriers include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions or emulsions areadministered orally, the active ingredient can be suspended or dissolvedin an oily phase combined with emulsifying or suspending agents. Ifdesired, certain sweetening, flavoring, or coloring agents can be added.

A nasal aerosol or inhalation composition can be prepared according totechniques well known in the art of pharmaceutical formulation. Forexample, such a composition can be prepared as a solution in saline,employing benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons, and/or othersolubilizing or dispersing agents known in the art. A composition havingone or more of the above-described compounds can also be administered inthe form of suppositories for rectal administration.

A pharmaceutically acceptable carrier is routinely used with one or moreactive above-mentioned conjugates. The carrier in the pharmaceuticalcomposition must be “acceptable” in the sense that it is compatible withthe active ingredient of the composition (and preferably, capable ofstabilizing the active ingredient) and not deleterious to the subject tobe treated. One or more solubilizing agents can be utilized aspharmaceutical excipients for delivery of an above-mentioned compound.Examples of other carriers include colloidal silicon oxide, magnesiumstearate, cellulose, sodium lauryl sulfate, and D&C Yellow #10.

The examples below are to be construed as merely illustrative, and notlimitative of the remainder of the disclosure in any way whatsoever.Without further elaboration, it is believed that one skilled in the artcan, based on the description herein, utilize the present invention toits fullest extent. All publications cited herein are herebyincorporated by reference in their entirety.

EXAMPLE 1 IFN-β-Di-PEG Polymer Conjugate Preparation of Di-PEG Aldehyde

20 kD PEGO(C═O)OSu was prepared from 20 kD mPEGOH purchased from (SunBioInc., CA, USA) according to the method described in Bioconjugate Chem.1993, 4, 568-569.

A solution of 6-(1,3-dioxolan-2-yl)hexane-1,5-diamine in dichloromethane(11.97 g of the solution containing 9.03 mg of diamine, 47.8 μmol) wasadded to a flask containing 20 kD PEGO(C═O)OSu (1.72 g, 86.0 μmol).After PEGO(C═O)OSu was completely dissolved, N, N-diisopropylethylamine(79 μL, 478 μmol) was added. The reaction mixture was stirred at roomtemperature for 24 h, and then methyl t-butyl ether (200 mL) was addeddropwise with stirring. The resulting precipitate was collected anddried under vacuum to give di-PEG acetal (1.69 g, 98%) as a white solid.

¹H NMR (400 MHz, d₆-DMSO) δ 7.16 (t, J=5.2 Hz, 1H), 7.06 (d, J=8.8 Hz,1H), 4.76 (t, J=4.8 Hz, 1H), 4.10-3.95 (m, 4H), 1.80-1.65 (m, 1H),1.65-1.50 (m, 1H), 1.48-1.10 (m, 6H).

Di-PEG acetal (4.0 g, 0.2 mmol) was suspended in pH 2.0 buffer (criticacid, 40 mL). The reaction mixture was stirred at 35° C. for 24 h andthen extracted with dichloromethane (3×50 mL). The combined organiclayers were dried over magnesium sulfate, concentrated, and thenre-dissolved in dichloromethane (20 mL). The solution was addeddropwisely to methyl t-butyl ether (400 mL) with stirring. The resultingprecipitate was collected and dried at reduced pressure to give di-PEGaldehyde (3.8 g, 95%) as a white solid.

¹H NMR (400 MHz, d₆-DMSO) δ 9.60 (s, 1H), 7.24 (d, J=8.4 Hz, 1H), 7.16(t, J=5.2 Hz, 1H), 4.10-3.95 (m, 4H), 3.95-3.80 (m, 1H), 3.00-2.85 (m,2H), 2.58-2.36 (m, 2H), 1.46-1.15 (m, 6H).

Alternatively, di-PEG aldehyde was prepared in the following manner:

The two amino groups of commercial available homo-lysine (Astatech

Pharmaceutical Co., Ltd, China) were protected by benzyloxycarbonyl. TheN-protected homo-lysine was esterified and reduced to form an aldehydecompound. The aldehyde group was subsequently protected with ethyleneglycol. The benzyloxycarbonyl protecting group then was removed byhydrogenation in the presence of a palladium catalyst. The N-deprotectedcompound was reacted with activated mPEGOH (Sunbio Chemicals Co., Ltd.,South Korea) in a mild basic condition. The resulting product wasstirred in pH 2.0 citric acid buffer (Sigma-Aldrich, Germany) at 25° C.for 72 hours to remove the aldehyde protecting group. 109 g of di-PEGpolymer aldehyde was obtained (yield: 95%). Purity was more than 97.7%(determined by HPLC) and more than 95% (determined by ¹H NMR analysis).

Preparation of Human rhIFN-β Ser₁₇

A DNA fragment encoding human INF-β Ser₁₇ was cloned into expressionvector pET24a to produce an expression plasmid rhIFN-β Ser₁₇-pET24a.This expression plasmid was transformed into E. coli and positivetransformants, i.e., clones carrying the expression plasmid, wereselected, cultivated, and the resultant E. coli cultures were stored at−80° C.

10 μl of a stored E. coli culture mentioned above were inoculated into200 ml of a seeding medium consisting of Terrific Broth and glycerol,for about 15 hours at 37° C. and 200 rpm. 150 ml of the E. coli culturethus obtained were transferred to 2.5 L culture medium containingglucose (10 g/L), MgSO₄.7H₂O (0.7 g/L), (NH₄)₂HPO₄ (4 g/L), KH₂PO₄ (3g/L), K₂HPO₄ (6 g/L), citrate (1.7 g/L), Yeast Extract (10 g/L),kanamycin (50 mg/ml), chloramphenicol (50 mg/ml), an antifoaming agent,and trace elements including FeSO₄.7H₂O (10 mg/L), ZnSO₄.7H₂O (2.25mg/L) CuSO₄.5H₂O (1 mg/L), MnSO₄.H₂O (0.5 mg/L), H₃BO₃ (0.3 mg/L),CaCl₂.2H₂O (2 mg/L), (NH₄)₆Mo₇O₂₄ (0.1 mg/L), EDTA (0.84 mg/L), and Cl(50 mg/L), and cultivated at 37° C. When the OD₆₀₀ of the E. coliculture reached 120 to 140, IPTG (1 M) was added to the culture toinduce expression of rhIFN-β Ser₁₇. The induced culture was incubated at37° C. and 300 rpm for 3 hours. When necessary, a feeding mediumcontaining 800 g/l glucose and 20 g/L MgSO₄ was added to the E. coliculture during incubation.

The E. coli culture obtained as described above was subjected tocentrifugation to harvest E. coli cells. The cells were resuspended in aPBS buffer (0.1M Na2HPO4, 0.15M NaCl) and disrupted in an APVHomogenizer. The homogenized solution thus obtained was centrifuged at10,000 rpm, 4° C. for 15 min. The precipitates (including inclusionbody) were collected, resuspended in PBS, and stirred at roomtemperature for 20-30 min to form a suspension. NaOH (6 N) was added tothe suspension to adjust its pH to 12 to allow dissolution of proteinsincluded in the inclusion body. About 2 minutes later, the pH value ofthe suspension was adjusted to 7.5 with 6 N HCl. The suspension was thensubjected to centrifugation and the supernatant thus formed wascollected, its protein concentration being determined using aspectrophotometer. The supernatant was mixed with a refolding buffer(TEA, pH 8.3) and incubated at room temperature without being stirredfor 24˜48 hours. It was then concentrated and dialyzed, using the TFFsystem and PLCCC cassette provided by Millipore, Inc. The resultantsolution was subjected to ultrafiltration, dialysis, and fractionationwith a SPFF Sepharose column. Fractions A9 and A10 thus obtained,containing the recombinant protein rhIFN-β Ser₁₇, were furtherfractionated with another SPFF Sepharose column to enrich therecombination protein (in Fractions A8-A10). TheserhIFN-βSer₁₇-containing fractions were further purified by gelfiltration (Superdex 75 HR 10/300) to obtain the rhIFN-β Ser₁₇ protein(1 mg/ml) having a purity of greater than 90%. The bioactivity of therecombinant protein was >2×10⁷ IU/mg protein.

Preparation of IFN-β-Di-PEG Polymer Conjugate

18.9 mg rhINF-β Ser₁₇ and 1.51 g diPEG aldehyde were suspended in 26 mLof 0.1 M sodium phosphate buffer (pH 5.0). To this solution was added400 eq. of NaCNBH₃ (Acros Organics, Belgium). The reaction mixture wasstirred at room temperature for 16 hours and then subjected to dialysiswith 25 mM tris-HCl (pH 7.8). The crude product was purified by anion-exchange column to afford 2 mg of IFN-β-di-PEG polymer.

Preparation of Human IFN-β

Transformed E. coli BLR (DE3)-RIL cells, carrying the encoding sequenceof IFN-β operatively linked to an E. coli promoter, were inoculated in250 mL SYN medium (10 g/L of select soytone, 5 g/L Yeast extract, and 10g/L NaCl) supplemented with 50 μl/mL kanamycin and 50 μl/mLchloramphenicol. The cells were then cultured at 37° C. in a shakerincubator at 220 rpm overnight (i.e., 16 hours).

250 mL of the overnight culture mentioned above were inoculated into 3.0L basic medium (10 g/L of Glucose, 0.7 g/L of MgSO₄.7H₂O, 4 g/L of(NH₄)₂HPO₄, 3 g/L of KH₂PO₄, 6 g/L of K₂HPO₄, 2 g/L of Citrate, 10 g/Lof Yeast extract and 2 g/L of Isoleucine) supplemented with 10 g/L basicglucose, 0.7 g/L feeding MgSO₄, 30 mL feeding trace element (10 g/L ofFeSO₄.7H₂O, 2.25 g/L of ZnSO₄.7H₂O, 1 g/L of CuSO₄. 5H₂O, 0.5 g/L ofMnSO4.H₂O, 0.3 g/L of H₃BO₃, 2 g/L of CaCl₂.2H₂O, 0.1 g/L of(NH4)₆Mo₇O₂₄, 0.84 g/L of EDTA, 50 ml/L of HCl), 25 μl/mL kanamycin and25 μl/mL chloramphenicol and cultured in a five liter fermentor (Bioflo3000; Brunswick Scientation Co., Edison N.J.). During fermentation, thepH of the medium was controlled at pH 7.1 by automated addition of a 37%NH₄OH solution. The dissolved oxygen (DO) level was maintained at 30%.The feeding solution (800 g/L of glucose, 20 g/L of MgSO₄, 50 μl/mLkanamycin and 50 μl/mL chloramphenicol) was added using aprogram-controlled pump, which was set to feed when DO level exceeds40˜60. When the cell density (OD₆₀₀) in the fermentation culture reached180 to 200, 4 mL of 1 M Isopropyl-β-D-1-thiogalactopyranoside (IPTG) wasadded to the fermentation culture to induce IFN β expression, togetherwith 30 mL of feeding trace elements and 25 g of yeast extract. Cellswere harvested 5 hours after IPTG induction by centrifugation.

The cell pellets were suspended in PBS buffer (0.1M sodium phosphate,0.15M sodium chloride, pH 7.4) at an approximate ratio of 1:3 (wetweight g/mL), disrupted by a microfluidizer, and then centrifuged at10,000 rpm for 20 min at 4° C. The pellet containing inclusion body (IB)was washed twice with PBS buffer, centrifuged as described above, andsuspended in 1 L PBS solution (0.1M sodium phosphate, 0.15M sodiumchloride, pH 7.4, 3% zwittergent 3-14, 5 mM DTT). After being stirredfor 30 minutes, the suspension was subjected to pH adjustment to 12 with6.0 M NaOH, while stirring to solubilize the pellet. The pH of thesuspension was then adjusted to pH 7.5 with 6 N HCl. Upon centrifugationat 10,000 rpm for 20 min, the supernatant, containing soluble IFN β, wascollected.

The soluble INF-β was then subjected to refolding as follows. Thesupernant mentioned above was diluted in 10 L of a freshly preparedrefolding buffer (100 mM Tris-HCl (pH 7.6), 0.5 M L-Arginine, 2 mM EDTA)for form a refolding mixture. The mixture was incubated for 48 hrwithout stirring. After incubation, the mixture, containing refoldedrecombinant IFN-β, was dialyzed against 20 mM Tris (with 100 mM NaCl,0.05% zwittergent 3-14, pH 7.0) buffer.

The dialyzed mixture was loaded onto a SP-Sepharose column (GE AmershamPharmacia), which was pre-equilibrated and washed with a 20 mM Tris-HCl,100 mM NaCl buffer (pH 7.0). IFN β was eluted with a solution containing20 mM Tris-HCl buffer (pH 7.0) and 200 mM NaCl. Fractions containing IFNβ was collected based on their absorbance at 280 nm. The IFN β containedtherein was further purified by a hydrophobic interaction column (GEhealthcare, Butyl Sepharose Fast Flow), which was pre-equilibrated andwashed with a solution containing1.0 M ammonium sulphate, 20 mM sodiumacetate and 0.05% zwittergent (pH 4.5). IFN β was eluted using asolution containing 0.5 M ammonium sulphate and 20 mM sodium acetate.Fractions containing the protein were collected based on theirabsorbance at 280 nm. These fractions were pooled and the concentrationof IFN β was determined by BCA protein assay (BCA™ Protein assay,Pierce).

Preparation of PEG-IFN-β Conjugate

To a solution of di-PEG aldehyde (296 mg, 7.4 μmol) in water (1.46 mL)was added 2 M sodium phosphate buffer (pH 4.0, 0.37 mL), zwittgen 3-14(1.48 mL, 10% in water) and INF-β (14.8 mg in 3.7 mL of pH 4.5 buffercontaining 20 mM sodium acetate, 0.7% ammonium sulfate and 0.05%detergent). The reaction mixture was stirred at room temperature for 10minutes, followed by addition of the cyanoborohydride aqueous solution(400 mM, 92.5 μL, 37 μmol). The reaction mixture was stirred in the darkfor 40 hours and purified by SP HP Sepharose chromatography. Fractionscontaining the desired PEG-IFN β conjugate were collected based on theirretention time and absorbance at 280 nm. The concentration of theconjugate was determined by BCA protein assay (BCA™ Protein assay,Pierce).

Pharmacokinetic Study in Rats

A pharmacokinetic study was performed in a rat model to compare serumhalf-life of IFN-β and PEG-IFN-β. Male rats (250˜350 gm) wereadministered intravenously at a dose of 600 μg/kg IFN β (n=3) andPEG-IFN β (n=3). Blood (250 μL) was collected from each rat beforeadministration and at 0.1, 1, 2, 4, 6, 10, 24, 48, 72, and 96 hoursafter administration. Serum samples were prepared from the blood and theamounts of IFN-β contained in the samples were analyzed by anEnzyme-linked immunoassay (ELISA). The serum half-life of IFN-β andPEG-IFN-β was 2 hours and 20 hours respectively, calculated from theserum concentration of the last three time points.

EXAMPLE 2 EPO-PEG Polymer Conjugate Preparation of PEG-EPO

To a solution of di-PEG aldehyde (267 mg, 6.1 μmol) in water (2.67 mL)was added 2 M sodium phosphate buffer (pH 4.0, 1 mL) and EPO (10 mg in3.03 mL of pH 7.3 buffer containing 20 mM sodium phosphate and 150 mMNaCl). The reaction mixture was stirred at room temperature for 10minutes, followed by the addition of the Sodium cyanoborohydride aqueoussolution (400 mM, 100 μL, 40 μmol). The reaction mixture was stirred inthe dark for 17 hours and purified by a SP Toyopearl column (Tosoh). Thecolumn was equilibrated with 20 mM Sodium acetate buffer, pH 4.5. Thereaction mixture was diluted to a concentration of 0.3-0.4 mg/ml andloaded onto the SP Toyopearl column. Fractions containing the desiredPEG-EPO conjugate were collected based on their retention time andabsorbance at 280 nm. The concentration of the conjugate was determinedby 280 nm UV absorbance.

Pharmacokinetic Study in Rats

A pharmacokinetic study was performed in a rat model to compare serumhalf-life of EPO and PEG-EPO. Male rats (250˜350 gm) were administeredintravenously with EPO (n=5) and PEG-EPO (n=5) at a dose of 25 μg/kg.Blood (250 μL) was drawn from each rat before administration and 0.088,0.75, 1.5, 3, 6, 10, 24, and 48 hours post administration. For PEG-EPOtreated rats, blood samples were further collected at 72 and 96 hoursafter administration. Serum samples were prepared from the blood andanalyzed with an Enzyme-linked immunoassay (ELISA) to determine theamounts of EPO contained therein. The results show that the serumhalf-life of EPO was 9 hours while that of PEG-EPO was significantlyincreased, i.e., 38 hours.

Preparation of EPO-PEG Polymer Conjugate

0.2 mg of EPO (Cashmere Scientific Company, Taiwan) and 4 mg of di-PEGaldehyde (20 equal.) were suspended in 0.1 M phosphate buffer (pH 5.0).To this solution was added 400 eq. of NaCNBH₃. The reaction mixture wasstirred at room temperature for 16 hours. HPLC confirmed formation ofEPO-di-PEG polymer.

EXAMPLE 3 GH-PEG Polymer Conjugate Preparation of Met-hGH

Transformed E. coli BLR (DE3)-RIL cells, capable of expression Met-hGH,were cultured following the fermentation procedure described above forexpression of Met-hGH.

Cells were harvested via centrifugation and cell pellet was suspended inTE buffer (50 mM Tris-HCl, 1 mM EDTA, pH 8.0) at an approximate ratio of1:3 (wet weight g/mL). The cells were then disrupted by a microfluidizerand then centrifuged at 10,000 rpm for 20 min. The pellet containinginclusion body (IB) was washed twice with TED buffer (50 mM T ris-HCl, 1mM EDTA, 2% Deoxycholate, pH 8.0), centrifuged as described above, andsuspended in MilliQ water and centrifuged at 20,000 rpm for 15 min. TheIBs were suspended in 400 mL of 50 mM TUD solution (50 mM Tris-HCl, 4 MUrea, 2.5 mM DTT, pH 10.0) and the suspension was centrifuged at 20,000rpm for 20 min; supernatant collected.

The supernatant was diluted in 2.0 L of a freshly prepared refoldingbuffer (50 mM Tris-HCl, 0.5 mM EDTA, 5% glycerol 10 mM GSH/1 mM GSSG, pH8.0). The mixture thus formed was incubated for 36 hr without stirringand then dialyzed against 20 mM Tris buffer (pH 7.0).

The dialyzed mixture, containing Met-hGH, was loaded onto a Q-Sepharosecolumn (GE Amersham Pharmacia, Pittsburgh, Pa.), which waspre-equilibrated and washed with a 20 mM Tris-HCl buffer, pH 7.0.Met-hGH was eluted a solution containing 20 mM Tris-HCl buffer, pH 7.0and 100 mM NaCl. Fractions containing Met-hGH, determined by theirabsorbance at 280 nm, were collected, pooled, and loaded onto ahydrophobic interaction column (GE Amersham Pharmacia, Pittsburgh, Pa.),pre-equilibrated and washed with a 20 mM sodium acetate buffer (pH 7.0),at a flow rate of 5 ml/min. Met-hGH was eluted with a solutioncontaining 20 mM sodium acetate buffer and 150 mM ammonium sulfate. Afraction containing Met-hGH was collected and subjected to BCA proteinassay (BCA™ Protein assay, Pierce) to determine the Met-hGHconcentration.

Preparation of PEG-Met-hGH Conjugate

To a solution of di-PEG aldehyde (74 mg, 1.7 μmol) in water (387 μL) wasadded 2 M sodium phosphate buffer (pH 4.0, 374 μL) and human GH (22.4 mgin 6.5 mL of pH 4.5 buffer containing 20 mM sodium acetate and 150 mMNaCl). The reaction mixture was stirred at room temperature for 10minutes, followed by the addition of the sodium cyanoborohydride aqueoussolution (400 mM, 140 μL, 56 μmol). The reaction mixture was stirred inthe dark for 17 hours and purified by SP XL Sepharose chromatography.Fractions containing the desired polymer-protein conjugate werecollected based on their retention time and absorbance at 280 nm. Theconcentration of conjugate was determined by a protein assay kit usingthe Bradford method (Pierce, Rockford, Ill.).

Pharmacokinetic Study in Rats

A pharmacokinetic study was performed in a rat model to compare serumhalf-life of Met-hGH and PEG-Met-hGH. Male rats (250˜350 gm) wereadministered intravenously with Met-hGH (n=5) or PEG-Met-hGH (n=5) at adose of 100 μg/kg. Blood samples were collected from Met-hGH-treatedrats before administration and 0.083, 1, 2, 4, 8, 12, and 24 hours afteradministration; and were collected from PEG-Met-hGH-treated rats beforeadministration and 0.33, 1, 4, 8, 12, 24, 48, 72, and 96 hours afteradministration. Serum samples were prepared from the blood and analyzedwith an Enzyme-linked immunoassay (ELISA) to determine hGHconcentrations. The serum half-life of Met-hGH and PEG-Met-hGH was 3hours and 35 hours respectively.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the scope of thefollowing claims.

1. A peptide-polymer conjugate of the following formula:

wherein each of R₁, R₂, R₃, R₄, and R₅, independently, is H, C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteraryl, C₃₋₈ cycloalkyl,or C₃₋₈ heterocycloalkyl; each of A₁ and A₂, independently, is a polymermoiety; each of G₁, G₂, and G₃, independently, is a bond or a linkingfunctional group; P is an erythropoietin moiety, the nitrogen atom ofthe N-terminus of P being bonded to G₃; m is 0 or an integer of 1-10;and n is an integer of 1-10.
 2. The conjugate of claim 1, wherein P isan erythropoietin moiety in which 1-4 additional amino acid residues areattached to the N-terminus of a native erythropoietin.
 3. The conjugateof claim 1, wherein each of A₁ and A₂ is a polyethylene glycol moietyhaving a molecular weight of 2-100 kD.
 4. The conjugate of claim 3,wherein each of A₁ and A₂ is a polyethylene glycol moiety having amolecular weight of 10-30 kD.
 5. The conjugate of claim 4, wherein eachof G₁ and G₂ is

in which the O is bonded to A₁ or A₂, and the N atom is bonded to acarbon atom; and G₃ is a bond.
 6. The conjugate of claim 5, wherein m is4, n is 2, and each of R₁, R₂, R₃, R₄, and R₅ is H.
 7. The conjugate ofclaim 1, wherein the conjugate is

in which mPEG is a methoxy-capped polyethylene glycol moiety having amolecular weight of 20 kD.
 8. A peptide-polymer conjugate of thefollowing formula:

wherein each of R₁, R₂, R₃, and R₄, independently, is H, C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteraryl, C₃₋₈ cycloalkyl, or C₃₋₈heterocycloalkyl; n is an integer of 2-10; A is a polymer moiety; G is alinking functional group; and P is an erythropoietin moiety, thenitrogen atom of the N-terminus of the peptide moiety being bonded tothe carbon atom in the

moiety shown in the formula above.
 9. The conjugate of claim 8, whereinG is

in which the O atom is bonded to A and the N atom is bonded to a carbonatom.
 10. The conjugate of claim 9, wherein A is a polyethylene glycolmoiety having a molecular weight of 10-40 kD.
 11. The conjugate of claim10, wherein A is a polyethylene glycol moiety having a molecular weightof 20-30 kD.
 12. The conjugate of claim 8, wherein n is 1.